Its. Eighteen selected strains have been assessed for siderophore production according to
Its. Eighteen selected strains were assessed for siderophore production based on the O-CAS method [17]. Phosphate-solubilizing activity was tested on Pikovskaya AMPA Receptor custom synthesis medium [18], NBRIP medium [19] and modified Burk’s agar medium [1], adding 0.5 of Ca3 (PO4 )two to every medium as insoluble P source. In both assays, Pseudomonas fluorescens2. Supplies and Methods2.1. Soil Sampling, Caspase 8 custom synthesis bacterial Isolation, and Azotobacter Reference Strains. In total, 74 bulk soil samples (00 cm) had been collected from agricultural (53 samples) and non-agricultural sites (21 samples) in the course of spring 2006. Samples belonged to 38 distinctive locations of Northwest, Pampas, and Patagonia regions of Argentina (see Supplementary Material out there online at dx.doi.org/10.1155/2013/519603). Soil aggregates (2 mm) had been spread onto the surface of Petri dishes containing N-free Burk’s agar medium with mannitol as C-source [1]. Just after 5 days at 28 C, slimy and glistening Azotobacter-like colonies growing around soil particles were chosen and further purified in N-free LG with bromothymol blue agar medium [1]. Motility, pigment production, and encystment had been determined as previously described [1].The Scientific Planet Journal BNM233 (Banco Nacional de Microorganismos, Buenos Aires, Argentina) was utilized as a positive handle. Auxin production was determined making use of a colorimetric assay [20], with measurements immediately after 1, two, three, and five days of growth in modified LG (LGSP) liquid medium containing 1 sucrose and 0.5 soymeal peptone. At every time interval, the number of cells (cfu mL-1 ) was determined by plate counting on LG agar. Nitrogenase activity was estimated by the acetylene reduction assay. Bacterial cultures were grown in N-free Burk’s agar medium at 28 C for 24 h and ethylene production was measured by gas chromatography [21], utilizing a Hewlett Packard Series II 5890 equipped having a flame ionization detector (FID) plus a stainless-steel Porapak N column (three.two mm 2 m; 80/100 mesh). The injector, oven, and detector temperatures were 110 C, 90 C, and 250 C, respectively. N2 was utilized as carrier gas (four.five cm s-1 linear gas velocity). Total protein concentration of bacterial cells was determined by the Lowry strategy together with the DC Protein Assay kit (BioRad, USA). Nitrogenase activity was expressed as mmol ethylene made per mg of protein in 24 h. Indole-3-acetic acid (IAA), gibberellic acid (GA3 ), and zeatin (Z) production had been determined for six chosen Azotobacter spp. strains grown in LGSP liquid medium at 28 C for 8 days. Z was identified and quantified by HPLC-UV, whereas IAA and GA3 had been identified by gas chromatography-mass spectrometry with selective ion monitoring (GC-MS-SIM), as previously described [21]. two.7. Effects of Azotobacter Inoculation and IAA Pure Solutions on the Variety of Seminal Roots and Root Hairs of Wheat Seedlings. For plant tests, seeds of wheat (Triticum aestivum cv. Baguette Premium 13, Nidera, Buenos Aires, Argentina) were surface-disinfected (1 NaClO for 3 minutes) and germinated in plastic containers (15 25 four cm) on filter paper soaked with sterile distilled water. To preserve humidity, containers have been wrapped in transparent plastic bags and placed inside a growth chamber at 25 C using a 16 h light/8 h dark regime for 24 h. For inoculation, bacterial strains had been grown in LGSP liquid medium at 28 C for eight days (108 cfu mL-1 ). Fifteen pregerminated seeds had been inoculated with one hundred L of bacterial culture (107 cells) per seed and grown for eight days as described ab.
